PROJECT SUMMARY While the genetic basis of craniofacial malformations is being discovered, we do not understand why phenotypic variation occurs, especially when caused by the same mutation. One example is holopresencephaly (HPE), a common disease that affects 1 in 250 conceptions, but only 1 in 10,000 live births due to intrauterine lethality. HPE is unique in that malformations range from mild facial hypotelorism to cyclopia in humans. Mutations in Sonic hedgehog (SHH), and other members of this pathway, cause HPE in some patients. However, mouse models of Shh mutations do not recreate the spectrum of phenotypes observed in patients. The objective of this proposal is to determine the mechanisms that cause facial shape variation in a novel mouse model of HPE that produces a full spectrum of mild to severe HPE phenotypes, and to determine the extent to which these outcomes can be reversed to approximate a more normal phenotype. Previous research in our lab has determined that signaling molecules (i.e., FGF8 and SHH) have a non-linear relationship between phenotypic variation and gene dosage. Specifically, this indicates that there is a threshold effect where small changes in gene dosage produce little to no effect, but produces wide variation below a threshold. Additionally, the downstream effects of altering gene dosage only alter downstream targets specific to the signaling pathway. Here, I propose to use a novel mouse model that has inactivation of nitric oxide synthase interacting protein (NOSIP). This mutation affects key developmental events through changes in mono-ubiquitination of protein phosphatase 2A (PP2A), which appear to affect SHH and Wnt signaling. In this work, I will quantify the genotype to phenotype map to provide novel insight on mechanisms that cause malformations and their variance. This application aims to test the hypothesis that inactivation of NOSIP produces a continuous spectrum of midfacial phenotypes mimicking those observed in human patients with HPE. Further, I will assess mechanisms contributing to face shape variation. In Aim 1, I will characterize the NOSIP mutants by quantifying face shape, Shh and Wnt signaling, gene expression levels, and cellular processes (proliferation, apoptosis, polarity). This results from Aim 1 will not only provide important mechanistic insight to the cause of general phenotypic variation, and the key cellular processes involved, but will also determine if the changes are due to specific pathway dysregulation or a more global effect. Aim 2 will determine the extent to which altering direct and indirect targets of NOSIP activity (i.e., PP2A, SHH, and Wnt) can reduce the phenotypic variance of Nosip mutants and restore a more normal phenotype. This aim will provide specific insight to the contribution of each regulator towards phenotypic variation, as well as possible therapeutic targets for HPE. Collectively, the two Aims will add significantly to our understanding of how phenotypic variation is produced and may provide clues for developing future therapeutic studies.